Our ongoing analysis of Nodal signaling involves the use of conditional mutagenesis: we have generated a conditional "floxed" allele, which is being used in conjunction with transgenic lines expressing Cre recombinase in various lineages of the early embryo. The T-Cre line developed by our colleague Mark Lewandoski at NCI-Frederick expresses Cre recombinase broadly in the mesodermal and definitive endodermal lineages commencing soon after gastrulation initiates. We found that T-Cre activity leads to complete deletion of the Nodal floxed allele prior to the normal onset of Nodal gene expression at the 3 to 4 somite stage in the left lateral plate mesoderm (LPM), the expression domain implicated in LR patterning. In embryos where both Nodal alleles are deleted in the left LPM we found a suite of LR defects, including right isomerisms of the lungs and reversed heart looping. Some deletion of the Nodal floxed allele was found also around the node. However, the level of expression was still close to normal at the late pre-somite stage, when asymmetric signaling from the node to the LPM is first established. In addition, the deleted allele was expressed more strongly on the left side of the node, the same asymmetric pattern seen for the wild type allele in normal embryos. Other node and midline markers showed normal patterns of expression as well, arguing that the remaining levels of Nodal at the node were likely above the critical threshold required to establish asymmetric signaling from the midline. Thus, the observed defects following T-Cre mediated deletion of Nodal stem from loss of function within the LPM domain only, providing evidence that Nodal expression at the node has no other role than to elicit Nodal expression in the LPM. Because only LR defects were observed it appears that essential developmental functions of the Nodal signaling pathway, at least within the extensive mesodermal and endodermal domains marked by T-Cre activity, are complete by e8.5. Additional work on Nodal in our lab is addressing the epigenetic regulation of its expression. Histone modifications have been proposed to function as an epigenetic code independent from their role in ongoing chromatin processes, with trimethylated lysine 4 on histone H3 (H3K4me3) mainly found in active promoter regions and trimethylated lysine 27 (H3K27me3) marking genes for repression by Polycomb proteins. The presence of both these modifications in promoters of developmentally important genes in embryonic stem (ES) cells recently has been suggested to function as an epigenetic signal marking these genes for transcriptional activation later in development. In a major new effort in the laboratory, we have found evidence connecting the Nodal-Smad2/3 pathway with Polycomb de-repression. We have found that Nodal and Brachyury, a target of Wnt/&amp;#946;-catenin signaling in mesoderm formation but which we have now identified as a direct target also of the Nodal pathway, are both regulated by Polycomb repression. Both genes are bound by Polycomb proteins and show H3K27me3 repressive histone marks in the absence of Nodal signaling, or in the absence of Nodal and Wnt signals in the case of Brachyury. We further found that the Nodal-Smad2/3 signaling pathway directly engages the H3K27me3 demethylase Jmjd3 to counteract Polycomb repression at target loci. Physical interaction between Smads2/3 with Jmjd3, and recruitment of Jmjd3 to target genes is dependent on active signaling. In the absence of Polycomb function, however, target loci are expressed independent of Nodal signaling. These results identify Polycomb de-repression as a novel function of the Nodal-Smad2/3 pathway, and suggest this may be a primary role in stem cells and perhaps the early embryo.Our ongoing analysis of Nodal signaling involves the use of conditional mutagenesis: we have generated a conditional "floxed" allele, which is being used in conjunction with transgenic lines expressing Cre recombinase in various lineages of the early embryo. The T-Cre line developed by our colleague Mark Lewandoski at NCI-Frederick expresses Cre recombinase broadly in the mesodermal and definitive endodermal lineages commencing soon after gastrulation initiates. We found that T-Cre activity leads to complete deletion of the Nodal floxed allele prior to the normal onset of Nodal gene expression at the 3 to 4 somite stage in the left lateral plate mesoderm (LPM), the expression domain implicated in LR patterning. In embryos where both Nodal alleles are deleted in the left LPM we found a suite of LR defects, including right isomerisms of the lungs and reversed heart looping. Some deletion of the Nodal floxed allele was found also around the node. However, the level of expression was still close to normal at the late pre-somite stage, when asymmetric signaling from the node to the LPM is first established. In addition, the deleted allele was expressed more strongly on the left side of the node, the same asymmetric pattern seen for the wild type allele in normal embryos. Other node and midline markers showed normal patterns of expression as well, arguing that the remaining levels of Nodal at the node were likely above the critical threshold required to establish asymmetric signaling from the midline. Thus, the observed defects following T-Cre mediated deletion of Nodal stem from loss of function within the LPM domain only, providing evidence that Nodal expression at the node has no other role than to elicit Nodal expression in the LPM. Because only LR defects were observed it appears that essential developmental functions of the Nodal signaling pathway, at least within the extensive mesodermal and endodermal domains marked by T-Cre activity, are complete by e8.5. Additional work on Nodal in our lab is addressing the epigenetic regulation of its expression. Histone modifications have been proposed to function as an epigenetic code independent from their role in ongoing chromatin processes, with trimethylated lysine 4 on histone H3 (H3K4me3) mainly found in active promoter regions and trimethylated lysine 27 (H3K27me3) marking genes for repression by Polycomb proteins. The presence of both these modifications in promoters of developmentally important genes in embryonic stem (ES) cells recently has been suggested to function as an epigenetic signal marking these genes for transcriptional activation later in development. In a major new effort in the laboratory, we have found evidence connecting the Nodal-Smad2/3 pathway with Polycomb de-repression. We have found that Nodal and Brachyury, a target of Wnt/&amp;#946;-catenin signaling in mesoderm formation but which we have now identified as a direct target also of the Nodal pathway, are both regulated by Polycomb repression. Both genes are bound by Polycomb proteins and show H3K27me3 repressive histone marks in the absence of Nodal signaling, or in the absence of Nodal and Wnt signals in the case of Brachyury. We further found that the Nodal-Smad2/3 signaling pathway directly engages the H3K27me3 demethylase Jmjd3 to counteract Polycomb repression at target loci. Physical interaction between Smads2/3 with Jmjd3, and recruitment of Jmjd3 to target genes is dependent on active signaling. In the absence of Polycomb function, however, target loci are expressed independent of Nodal signaling. These results identify Polycomb de-repression as a novel function of the Nodal-Smad2/3 pathway, and suggest this may be a primary role in stem cells and perhaps the early embryo.